Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability.

We cloned, purified and characterized two extremophilic cytidine deaminases: CDA(Bcald) and CDA(Bpsy), isolated from Bacillus caldolyticus (growth at 72 degrees C) and Bacillus psychrophilus (growth at 10 degrees C), respectively. We compared their thermostability also with the mesophilic counterpart, CDA(Bsubt), isolated from Bacillus subtilis (growth at 37 degrees C). The DNA fragments encoding CDA(Bcald) and CDA(Bpsy) were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDA(Bsubt). Both enzymes were found to be homotetramers of approximately 58 kDa. CDA(Bcald) was found to be highly thermostable, as expected, up to 65 degrees C, whereas CDA(Bpsy) showed higher specific activity at lower temperatures and was considerably less thermostable than CDA(Bcald). After partial denaturation at 72 degrees C for 30 min, followed by renaturation on ice, CDA(Bcald) recovered 100% of its enzymatic activity, whereas CDA(Bpsy) as well as CDA(Bsubt) were irreversibly inactivated. Circular dichroism (CD) spectra of CDA(Bcald) and CDA(Bpsy) at temperatures ranging from 10 to 95 degrees C showed a markedly different thermostability of their secondary structures: at 10 and 25 degrees C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50-70 degrees C, the alpha-helices of CDA(Bpsy) unfolded almost completely, whereas its beta-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDA(Bcald) with increase in temperature.

Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase.

The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactis strains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functional cdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations.

Deoxynucleoside kinases encoded by the yaaG and yaaF genes of Bacillus subtilis. Substrate specificity and kinetic analysis of deoxyguanosine kinase with UTP as the preferred phosphate donor.

The overlapping yaaG and yaaF genes from Bacillus subtilis were cloned and overexpressed in Escherichia coli. Purification of the gene products showed that yaaG encoded a homodimeric deoxyguanosine kinase (dGK) and that yaaF encoded a homodimeric deoxynucleoside kinase capable of phosphorylating both deoxyadenosine and deoxycytidine. The latter was identical to a previously characterized dAdo/dCyd kinase (Møllgaard, H. (1980) J. Biol. Chem. 255, 8216-8220). The purified recombinant dGK was highly specific toward 6-oxopurine 2'-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2',3'-dideoxyguanosine. UTP was the preferred phosphate donor with a Km value of 6 microm compared with 36 microm for ATP. In addition, the Km for dGuo was 0.6 microm with UTP but 6.5 microm with ATP as phosphate donor. The combination of these two effects makes UTP over 50 times more efficient than ATP. Initial velocity and product inhibition studies indicated that the reaction with dGuo and UTP as substrates followed an Ordered Bi Bi reaction mechanism with UTP as the leading substrate and UDP the last product to leave. dGTP was a potent competitive inhibitor with respect to UTP. Above 30 microm of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.

The role of zinc in Bacillus subtilis cytidine deaminase.

Cytidine deaminase (CDA) from Bacillus subtilis is a zinc-containing enzyme responsible for the hydrolytic deamination of cytidine to uridine and 2'-deoxycytidine to 2'-deoxyuridine. Titration of the cysteinyl groups of the enzyme with p-hydroxymercuriphenyl sulfonate (PMPS) resulted in release of one zinc ion per subunit. Addition of EDTA to chelate the zinc and dithiothreitol (DTT) to remove PMPS, followed by removal of the low molecular weight compounds by gel filtration, resulted in an apoenzyme with no enzymatic activity. The apoenzyme was almost fully reactivated by addition of zinc chloride, indicating that the zinc ion played a central role in catalysis, in keeping with what has been observed with Escherichia coli CDA [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., and Carter, C. W. J. (1994) J. Mol. Biol. 235, 635-656]. Addition of Cd(2+) or Co(2+) caused partial reactivation of the apoenzyme. Zinc reconstitution of the apoenzyme was strictly dependent on the presence of reducing agents, suggesting that the zinc-ligating cysteines, when unligated, participated in disulfide bond formation. An enzymatically active isoform of the tetrameric CDA protein, containing an extension of 13 amino acids at the C-terminus of each subunit, was used in conjunction with the wild-type CDA in subunit-subunit dissociation studies to show that the zinc ion does not assist in the thermodynamic refolding of the protein. After treatment with PMPS and EDTA, the enzyme existed as unfolded unassociated subunits. Immediately following DTT addition to remove PMPS, the subunits refolded into a tetrameric structure, independent of the presence of zinc.

Expression of human mitochondrial thymidine kinase in Escherichia coli: correlation between the enzymatic activity of pyrimidine nucleoside analogues and their inhibitory effect on bacterial growth.

Mitochondrial thymidine kinase (TK2) phosphorylates pyrimidine nucleosides to monophosphates and is expressed constitutively through the cell cycle in all cells. Because of the overlap of its substrate specificity with that of the cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), it has been difficult to determine the role of TK2 in activating nucleosides used in chemotherapy. In this report, we described the construction of a recombinant Escherichia coli strain which could be used to test if TK2 activity is limiting for the toxicity of nucleosides. Enzymes of bacterial origin which are involved in thymidine and deoxyuridine anabolism and catabolism were eliminated, and the cDNA for human TK2 was introduced. In the crude extract of the engineered E. coli, the level of thymidine kinase was, after induction of TK2 expression, several hundred fold higher than in the control strain. Several pharmacologically interesting nucleoside analogues, including 3'-azidothymidine, 2',3'-didehydro-2',3'-dideoxythymidine, and 2', 3'-dideoxy-beta-L-3'-thiacytidine, were tested for their effects on the growth of this recombinant strain. For a comparison, the phosphorylation of these compounds was determined with purified recombinant TK1, TK2, and dCK. A correlation was observed between the phosphorylation of several of these compounds by TK2 and their effects on bacterial growth. These results demonstrate that activation of growth-inhibiting pyrimidine nucleosides can be catalyzed by TK2, and together with recombinant E. coli strains expressing other cellular nucleoside kinases, this whole-cell bacterial system may serve as a tool to predict the efficacy and side effects of chemotherapeutic nucleosides.

Genetic and biochemical characterization of Salmonella enterica serovar typhi deoxyribokinase.

We identified in the genome of Salmonella enterica serovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between the uhpA and ilvN genes, is absent in Escherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. coli correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coli ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the V(max) of deoxyribokinase by a factor of 2.5 and increased the K(m) for deoxyribose by a factor of 70, compared to the parent enzyme.

Possible role of two phenylalanine residues in the active site of human cytidine deaminase.

Site-directed mutagenesis on human cytidine deaminase (CDA) was employed to mutate specifically two highly conserved phenylalanine residues, F36 and F137, to tryptophan; at the same time, the unique tryptophan residue present in the sequence at position 113 was mutated to phenylalanine. These double mutations were performed in order to have for each protein a single tryptophan signal for fluorescence studies relative to position 36 or 137. The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F, showed by circular dicroism and thermal stability an overall structure not greatly affected by the mutations. The titration of Trp residues by N-bromosuccinimide (NBS) suggested that residue W113 of the wild-type CDA and W36 of mutant F36W/W113F are buried in the tertiary structure of the enzyme, whereas the residue W137 of mutant F137W/W113F is located near the surface of the molecule. Kinetic experiments and equilibrium experiments with FZEB showed that the residue W113 seems not to be part of the active site of the enzyme whereas the Phe/Trp substitution in F36W/W113F and F137W/W113F mutant enzymes had a negative effect on substrate binding and catalysis, suggesting that F137 and F36 of the wild-type CDA are involved in a stabilizing interaction between ligand and enzyme.

Cytidine deaminases from B. subtilis and E. coli: compensating effects of changing zinc coordination and quaternary structure.

Cytidine deaminase from E. coli is a dimer of identical subunits (M(r) = 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtilis is a tetramer of identical subunits (M(r) = 14 800). After purification from an overexpressing strain, the enzyme from B. subtilis is found to contain a single atom of zinc per enzyme subunit by flame atomic absorption spectroscopy. Fluorescence titration indicates that each of the four subunits contains a binding site for the transition state analogue inhibitor 5-fluoro-3,4-dihydrouridine. A region of amino acid sequence homology, containing residues that are involved in zinc coordination in the enzyme from E. coli, strongly suggests that in the enzyme from B. subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) [Song, B. H., and Neuhard, J. (1989) Mol. Gen. Genet. 216, 462-468]. This pattern of zinc coordination appears to be novel for a hydrolytic enzyme, and might be expected to reduce the reactivity of the active site substantially compared with that of the enzyme from E. coli (His-102, Cys-129, and Cys-132). Instead, the B. subtilis and E. coli enzymes are found to be similar in their activities, and also in their relative binding affinities for a series of structurally related inhibitors with binding affinities that span a range of 6 orders of magnitude. In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit. Sequence alignments, together with model building, suggest one possible mechanism of compensation.

Ribosomal -1 frameshifting during decoding of Bacillus subtilis cdd occurs at the sequence CGA AAG.

During translation of the Bacillus subtilis cdd gene, encoding cytidine deaminase (CDA), a ribosomal -1 frameshift occurs near the stop codon, resulting in a CDA subunit extended by 13 amino acids. The frequency of the frameshift is approximately 16%, and it occurs both when the cdd gene is expressed from a multicopy plasmid in Escherichia coli and when it is expressed from the chromosomal copy in B. subtilis. As a result, heterotetrameric forms of the enzyme are formed in vivo along with the dominant homotetrameric species. The different forms have approximately the same specific activity. The cdd gene was cloned in pUC19 such that the lacZ' gene of the vector followed the cdd gene in the -1 reading frame immediately after the cdd stop codon. By using site-directed mutagenesis of the cdd-lacZ' fusion, it was shown that frameshifting occurred at the sequence CGA AAG, 9 bp upstream of the in-frame cdd stop codon, and that it was stimulated by a Shine-Dalgarno-like sequence located 14 bp upstream of the shift site. The possible function of this frameshift in gene expression is discussed.

Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana.

The complementary DNA (cDNA) coding for Arabidopsis thaliana cytidine deaminase 1 (AT-CDA1) was obtained from the amplified A. thaliana cDNA expression library, provided by R. W. Davis (Stanford University, CA). AT-CDA1 cDNA was subcloned into the expression vector pTrc99-A and the protein, expressed in Escherichia coli following induction with isopropyl 1-thio-beta-d-galactopyranoside, showed high cytidine deaminase activity. The nucleotide sequence showed a 903-bp open reading frame encoding a polypeptide of 301 amino acids with a calculated molecular mass of 32,582. The deduced amino acid sequence of AT-CDA1 showed no transit peptide for targeting to the chloroplast or mitochondria indicating that this form of cytidine deaminase is probably expressed in the cytosol. The recombinant AT-CDA1 was purified to homogeneity by a heat treatment followed by an ion-exchange chromatography. The final enzyme preparation was >98% pure as judged by SDS-PAGE and showed a specific activity of 74 U/mg. The molecular mass of AT-CDA1 estimated by gel filtration was 63 kDa, indicating, in contrast to the other eukaryotic CDAs, that the enzyme is a dimer composed of two identical subunits. Inductively coupled plasma-optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit. The kinetic properties of AT-CDA1 both toward the natural substrates and with analogs indicated that the catalytic mechanism of the plant enzyme is probably very similar to that of the human the E. coli enzymes.

A combination of three mutations, dcd, pyrH, and cdd, establishes thymidine (Deoxyuridine) auxotrophy in thyA+ strains of Salmonella typhimurium.

The dum gene of Salmonella typhimurium was originally identified as a gene involved in dUMP synthesis (C. F. Beck et al., J. Bacteriol. 129:305-316, 1977). In the genetic background used in their selection, the joint acquisition of a dcd (dCTP deaminase) and a dum mutation established a condition of thymidine (deoxyuridine) auxotrophy. In this study, we show that dum is identical to pyrH, the gene encoding UMP kinase. The level of UMP kinase activity in the dum mutant was found to be only 30% of that observed for the dum+ strain. Thymidine prototrophy was restored to the original dum dcd mutant (KP1361) either by transduction using a pyrH+ donor or by complementation with either of two pyrH+-carrying plasmids. Thymidine auxotrophy could be reconstructed in the dum+ derivative (KP1389) by the introduction of a mutant pyrH allele. To define the minimal mutational complement necessary to produce thymidine auxotrophy in thyA+ strains, a dcd::Km null mutation was constructed. In the wild-type background, dcd::Km alone or in combination with a pyrH (dum) mutation did not result in a thymidine requirement. A third mutation, cdd (cytidine-deoxycytidine deaminase), was required together with the dcd and pyrH mutations to impart thymidine auxotrophy.

An Escherichia coli system expressing human deoxyribonucleoside salvage enzymes for evaluation of potential antiproliferative nucleoside analogs.

Deoxyribonucleoside salvage in animal cells is mainly dependent on two cytosolic enzymes, thymidine kinase (TK1) and deoxycytidine kinase (dCK), while Escherichia coli expresses only one type of deoxynucleoside kinase, i.e., TK. A bacterial whole-cell system based on genetically modified E. coli was developed in which the relevant bacterial deoxypyrimidine metabolic enzymes were mutated, and the cDNA for human dCK or TK1 under the control of the lac promoter was introduced. The TK level in extract from induced bacteria with cDNA for human TK1 was found to be 20,000-fold higher than that in the parental strain, and for the strain with human dCK, the enzyme activity was 160-fold higher. The in vivo incorporation of deoxythymidine (Thd) and deoxycytidine (dCyd) into bacterial DNA by the two recombinant strains was 20 and 40 times higher, respectively, than that of the parental cells. A number of nucleoside analogs, including cytosine arabinoside, 5-fluoro-dCyd, difluoro-dCyd, and several 5-halogenated deoxyuridine analogs, were tested with the bacterial system, as well as with human T-lymphoblast CEM cells. The results showed a close correlation between the inhibitory effects of several important cytostatic and antiviral analogs on the recombinant bacteria and the cellular system. Thus, E. coli expressing human salvage kinases is a rapid and convenient model system which may complement other screening methods in drug discovery projects.

Identification of four amino acid residues essential for catalysis in human cytidine deaminase by site-directed mutagenesis and chemical modifications.

By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild-type CDA. Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest that Glu67 is essential for the catalytic process. Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according to the sequence data. When p-hydroxymercuriphenyl sulfonate was used, nine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2-pyridylazo)resorcinol. It seems plausible that the limiting step for the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.

Recombinant uracil phosphoribosyltransferase from the thermophile Bacillus caldolyticus: expression, purification, and partial characterization.

The upp gene encoding the major uracil phosphoribosyltransferase (UPRT) of the thermophile Bacillus caldolyticus was cloned by complementation of an Escherichia coli upp mutation. The nucleotide sequence of the cloned DNA revealed an open reading frame of 630 bp encoding a polypeptide of 209 amino acids (M(r) 22,817) with 84% amino acid sequence identity to the deduced upp gene product of Bacillus subtilis. Primer extension analysis indicated that the transcriptional start site of the cloned gene was positioned 37 or 38 bp upstream of the coding region. When over-expressed in E. coli, the recombinant UPRT represented approximately 18% of the soluble cellular proteins. The enzyme was purified to homogeneity by two sequential precipitations with 50 mM Na-phosphate, pH 7.0. Gel filtration chromatography indicated that the native enzyme existed as a dimer at high protein concentrations but that it dissociated to a monomeric form on dilution. In dilute solutions the enzyme is highly unstable but can be stabilized by addition of bovine serum albumin. In concentrated solution (> 5 mg/ml) the enzyme is stable for months at 4 degrees C, even in the absence of bovine serum albumin. By comparing the UPRT activity of crude extracts of B. subtilis and B. caldolyticus it was found that the enzyme from B. caldolyticus was considerably more stable toward thermal inactivation than the homologous enzyme from B. subtilis.

Structural and catalytic properties of CMP kinase from Bacillus subtilis: a comparative analysis with the homologous enzyme from Escherichia coli.

CMP kinases from Bacillus subtilis and from Escherichia coli are encoded by the cmk gene (formerly known as jofC in B. subtilis and as mssA in E. coli). Similar in their primary structure (43% identity and 67% similarity in amino acid sequence), the two proteins exhibit significant differences in nucleotide binding and catalysis. ATP, dATP, and GTP are equally effective as phosphate donors with E. coli CMP kinase whereas GTP is a poor substrate with B. subtilis CMP kinase. While CMP and dCMP are the best phosphate acceptors of both CMP kinases, the specific activity with these substrates and ATP as donor are 7- to 10-fold higher in the E. coli enzyme; the relative Vm values with UMP and CMP are 0.1 for the B. subtilis CMP kinase and 0.01 for the E. coli enzyme. CMP increased the affinity of E. coli CMP kinase for ATP or for the fluorescent analog 3'-anthraniloyl dATP by one order of magnitude but had no effect on the B. subtilis enzyme. The differences in the catalytic properties of B. subtilis and E. coli CMP kinases might be reflected in the structure of the two proteins as inferred from infrared spectroscopy. Whereas the spectrum of B. subtilis CMP kinase is dominated by a band at 1633 cm-1 (representing beta type structures), the spectrum of the E. coli enzyme is dominated by two bands at 1653 and 1642 cm-1 associated with alpha-helical and unordered structures, respectively. CMP induced similar spectral changes in both proteins with a rearrangement of some of the beta-structures. ATP increases the denaturation temperature of B. subtilis CMP kinase by 9.3 degrees C, whereas in the case of the E. coli enzyme, binding of ATP has only a minor effect.

Utilization of orotate as a pyrimidine source by Salmonella typhimurium and Escherichia coli requires the dicarboxylate transport protein encoded by dctA.

Mutants deficient in orotate utilization (initially termed out mutants) were isolated by selection for resistance to 5-fluoroorotate (FOA), and the mutations of 12 independently obtained isolates were found to map at 79 to 80 min on the Salmonella typhimurium chromosome. A gene complementing the mutations was cloned and sequenced and found to possess extensive sequence identity to characterized genes for C4-dicarboxylate transport (dctA) in Rhizobium species and to the sequence inferred to be the dctA gene of Escherichia coli. The mutants were unable to utilize succinate, malate, or fumarate as sole carbon source, an expected phenotype of dctA mutants, and introduction of the cloned DNA resulted in restoration of both C4-dicarboxylate and orotate utilization. Further, succinate was found to compete with orotate for entry into the cell. The S. typhimurium dctA gene encodes a highly hydrophobic polypeptide of 45.4 kDa, and the polypeptide was found to be enriched in the membrane fraction of minicells harboring a dctA+ plasmid. The DNA immediately upstream of the deduced -35 region contains a putative cyclic AMP-cyclic AMP receptor protein complex binding site, thus affording an explanation for the more effective utilization of orotate with glycerol than with glucose as carbon source. The E. coli dctA gene was cloned from a lambda vector and shown to complement C4-dicarboxylate and orotate utilization in FOA-resistant mutants of both E. coli and S. typhimurium. The accumulated results demonstrate that the dctA gene product, in addition to transporting C4-dicarboxylates, mediates the transport of orotate, a cyclic monocarboxylate.

Recombinant human cytidine deaminase: expression, purification, and characterization.

The complementary DNA (cDNA) coding for human cytidine deaminase (CDA) was obtained using two specific primers to screen RNA from peripheral blood polymorphonuclear leukocytes by reverse transcriptase PCR. The cDNA fragment was ligated into the expression vector pTrc99-A and expressed in Escherichia coli following induction with isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The nucleotide sequence of the cDNA corresponded to that published by Laliberté and Momparler (Cancer Res. 54, 5401-5407, 1994). It contained a 438-bp open reading frame encoding a polypeptide of 146 amino acids with a predicted molecular mass of 16.2 kDa. The protein expressed in E. coli showed high cytidine deaminase activity and its molecular mass was estimated to be 57 kDa by gel filtration and 16 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Both values are in agreement with those already published and suggest that human CDA contains three or four identical subunits. Cross-linking experiment indicated that the enzyme is a tetramer. The recombinant CDA has been purified to homogeneity by a rapid procedure consisting of heat inactivation followed by affinity chromatography. The final enzyme preparation showed a specific activity of 105 U/mg, corresponding to about 88-fold purification with respect to the crude extract and was judged to be >98% pure by SDS-PAGE. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis revealed the presence of 1 atom of Zn per subunit. Since CDA causes the deamination of several antitumoral cytidine-analog drugs, the recombinant enzyme was characterized kinetically and several pyrimidine nucleoside analogs were tested as potential substrates and inhibitors. The results obtained agreed closely with those previously reported for the purified human placenta CDA.